CN115097129A - Detection reagent composition for placenta growth factor and soluble fms-like tyrosine kinase-1 - Google Patents
Detection reagent composition for placenta growth factor and soluble fms-like tyrosine kinase-1 Download PDFInfo
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Abstract
The invention relates to a detection reagent combination for placenta growth factor and soluble fms-like tyrosine kinase-1. The invention provides a method for detecting the content of placenta growth factor and soluble fms-like tyrosine kinase-1 in blood based on quantum dot fluorescence, which can be used for diagnosing early-onset eclampsia of pregnant and lying-in women. Compared with the existing detection method, the method provided by the invention increases the washing step, improves the nonspecific adsorption of the target, amplifies the detection signal through the streptavidin-biotin amplification system, realizes a more sensitive detection effect, and has a higher clinical application value.
Description
Technical Field
The invention belongs to the technical field of blood marker detection, and particularly relates to a detection reagent combination and a detection method of placenta growth factor and soluble fms-like tyrosine kinase-1, and application of the detection reagent combination and the detection method in the field of diagnosis of early-onset eclampsia.
Background
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Early-onset preeclampsia (EOPE) belongs to a special type of hypertensive diseases in pregnancy, and occurs after 20 weeks of pregnancy and before 34 weeks of pregnancy, and clinical manifestations that blood pressure is rapidly increased and difficult to control, a large amount of proteinuria occurs, and target organ damage is combined to seriously affect the health of mother and infant. Early diagnosis and intervention treatment of EOPE are of great significance to reduce adverse maternal and infant outcome. The pathogenesis of EOEP is not completely clarified, and vascular endothelial injury and placental ischemia and hypoxia are generally considered to be related to the occurrence and development of EOPE at present. Soluble FMS-like tyrosine kinase-1 (sFIt-1) is an anti-angiogenic factor, can cause vascular endothelial injury and angiogenesis disorder, and obviously increases the sFIt-1 level in blood when pregnancy is abnormal, and can reflect the degree of functional injury of vascular endothelial cells. The placenta growth factor (PLGF) is one of the important proteins in pregnancy, belongs to the vascular endothelial growth factor family, and can promote the formation of placenta vascular network and ensure the growth and development of fetus. The research proves that: the levels of sFIt-1 and PLGF in the serum and urine of the pregnant women before eclampsia are higher than those of normal pregnant women.
Soluble fms-like tyrosine kinase-1 (sFIt-1) in the blood of pregnant women belongs to the type III receptor tyrosine kinase family, and the molecular weight is about 63.6 KD; the protein consists of 1-6 immunoglobulin-like structural domains of an extracellular region, does not contain a tyrosine kinase domain, has a coding region completely consistent with an extracellular region of fms-like tyrosine kinase-1 (sFIt-1), and can compete with a membrane surface receptor to combine with Vascular Endothelial Growth Factor (VEGF). sFIt-1 was originally found on umbilical vein-like cells as a direct inhibitor of VEGF and placental growth factor (PLGF), and elevated serum levels can lead to poor growth of placental trophoblasts and blood vessels, resulting in damage to the inner cells. sFIt-1 is mainly combined with VEGF and PLGF to mediate the biological functions of VEGF and PLGF, and participates in the formation, remodeling and replanting of blood vessels, so that the prediction of sFIt-1 and preeclampsia is the focus of research. Changes in the concentration levels of PLGF and sFIt-1 in the blood circulation can be used to identify normal pregnancy and preeclampsia before clinical symptoms appear; the PLGF and the sFIt-1 are the first biochemical markers which are verified by large-scale clinical tests and can be used for predicting and diagnosing preeclampsia of pregnant women.
At present, enzyme-linked immunosorbent assay (ELISA), time-resolved fluorescence immunoassay (TR-FIA), microfluidic, Electrochemiluminescence (ECL) and other reports exist for detecting PLGF; however, the detection of sFIt-1 is reported less, mainly by the Roche electrochemiluminescence method, and all the above methods are directed to the type of the sample, namely a serum sample.
Considering that the quantum dot immunofluorescence technology has high accuracy and simple and rapid operation, the compatibility of the quantum dot immunofluorescence technology with blood samples (whole blood, serum and plasma) greatly widens the clinical application range of the quantum dot immunofluorescence technology, can realize the follow-up diagnosis and is convenient for clinical application; and the quantum dot immunofluorescence technology can carry out joint detection on PLGF and sFIt-1 in blood, can simultaneously provide detection results of the concentration of the PLGF and the sFIt-1 and the ratio of the sFIt-1 to the PLGF, and has higher clinical application value. Based on the method, the quantum dot fluorescence detection method of the placenta growth factor and the soluble fms-like tyrosine kinase-1 has important clinical detection significance.
Disclosure of Invention
As described in the background, the concentration of placental growth factor (PLGF), soluble fms-like tyrosine kinase-1 (sFIt-1) in blood is of great interest for pregnant women. The existing detection method has the defects of limited samples or insufficient sensitivity and the like. In order to solve the technical problems, the invention provides a quantum dot fluorescence detection method of placenta growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1).
In a first aspect of the invention, a detection reagent combination for placenta growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1) is provided, wherein the detection reagent combination comprises a detergent and a test strip for cleaning a blood sample to be detected;
the detergent comprises serum, polyethylene glycol, a non-ionic surfactant and a bacteriostatic agent;
the test strip comprises a bottom plate, a chromatographic structure is covered on the bottom plate, a blood filtering membrane, a combination pad, a detection pad and water absorption paper are sequentially arranged on the chromatographic structure according to the sample flowing direction, and the adjacent parts of the above components are connected in an overlapping mode.
In the first aspect, a combination of reagents for simultaneously detecting placental growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1) is provided, wherein the reagent is used for performing quantum dot fluorescence detection on PLGF and sFIt-1 in a washed sample by using a blood sample as a detection object based on a double antibody sandwich method. Compared with the existing detection method, the detection method has the obvious difference that the washing step is added, and the washing agent is used for washing the sample after sample loading, so that the nonspecific combination of the blood sample in the subsequent chromatographic detection process can be effectively reduced, and the detection sensitivity is improved.
Preferably, the serum can adopt fetal calf serum, newborn calf serum or calf serum; in a further preferred embodiment, newborn bovine serum, including but not limited to advanced newborn bovine serum, standard newborn bovine serum or common newborn bovine serum is used.
Preferably, the molecular weight of the polyethylene glycol is 5000-25000, and the polyethylene glycol comprises a polyethylene glycol high polymer or a polyethylene glycol derivative; in specific examples, the polyethylene glycol can be PEG6000, PEG8000 or PEG 20000.
Preferably, the nonionic surfactant is selected from one or a combination of several of S17, S18, S19, S21, S22 and S24; more preferably, the nonionic surfactant is S17.
Preferably, the bacteriostatic agent is ProClin300 (CAS number 96118-96-6).
In addition, the detergent should also maintain a similar osmotic environment as the blood sample, using physiological saline and/or buffer as solvent; one specific example of the buffer is HEPES buffer; in one embodiment of the above preferred embodiment, the formulation of the detergent is as follows: 0.07-0.13M HEPES buffer (pH 7-8) contains 0.8-1.0% NaCl, 3-6% newborn bovine serum, 1-2% PEG20000, 0.3-0.6% S17 and 0.03-0.06% Proclin 300.
The test strip provided by the first aspect of the present invention is an immunochromatographic test strip, and considering that a whole blood sample contains 45% of blood cells, and the rupture or the flow of the blood cells (mostly red blood cells) through a window will affect the test result, so that the detection value has a large deviation, therefore, a blood filter membrane is added at the sample application hole end of the test strip (the blood filter membrane is pretreated), the red blood cells are intercepted, and the adverse effect on the detection result is reduced. The blood filter membrane contains anti-erythrocyte antibodies (RBC) and is used for filtering out erythrocytes in the whole blood sample; the pretreatment of the blood filtering membrane is prepared by spraying RBC pretreatment liquid, and the formula of the pretreatment liquid is as follows: 0.07-0.13M HEPES buffer solution (pH 7-8) contains 0.8-1.0% NaCl, 1-3% sodium caseinate, 1-3% PVP-K30, 0.4-1.0% S17, 0.03-0.06% Proclin300 and 140-160 mu g/mL anti-erythrocyte antibody (RBC), and the blood filtering membrane is obtained by spraying the pretreatment solution according to the formula and then drying; in order to retain the activity of RBC, the drying is preferably performed in a low-temperature drying mode, such as a freeze drying mode, and the research of the invention finds that the method of freeze drying after pre-spraying a blood filtering membrane can not only retain the activity of RBC to the maximum extent, but also can seal vacant sites on the blood filtering membrane, avoid adsorbing target substances (PLGF and sFIt-1) in a blood sample, and further achieve the purpose of improving the sensitivity.
In one embodiment, the test strip detects placenta growth factor (PLGF) and soluble fms-like tyrosine kinase-1 in a blood sample by quantum dot fluorescence; as the detection sensitivity of the target objects (PLGF and sFIt-1) to be detected is high, the detection ranges of the Roche kit serving as the contrast reagent are 3-10000pg/mL and 10-85000pg/mL respectively, and the lower detection limit of the quantum dot immunofluorescence method is about 0.02 ng/mL. In order to improve the detection sensitivity, a streptavidin-biotin amplification system is further introduced, a mixture of quantum dot labeled streptavidin (Q-SA), a biotin labeled PLGF antibody (Bio-Ab1) and a biotin labeled sFIt-1 antibody (Bio-Ab2) is coated on a binding pad, the mixture is dissolved and diluted by a re-solution and then sprayed on the binding pad, and the formula of the re-solution is as follows: 0.07-0.13M HEPES buffer solution (pH 7-8) contains 0.8-1.0% NaCl, 1-3% tyrosine, 4-6% sucrose, 1-2% beta-cyclodextrin, 0.3-0.6% S17 and 0.03-0.06% Proclin 300; in order to ensure the high activity and stability of the quantum dot labeled streptavidin and the biotin labeled antibody to the maximum extent, the bonding pad is sprayed and then dried in a low-temperature drying mode, such as freeze drying.
The detection pad is sequentially provided with a detection line T1, a detection line T2 and a quality control line along the chromatography direction, the detection line T1 is coated with a PLGF capture antibody, the detection line T2 is coated with an sFIt-1 capture antibody, and the quality control line is coated with a goat anti-mouse IgG antibody.
In the test strip, the blood filtering membrane, the combination pad and the detection pad are sequentially overlapped, and the part of the absorbent paper adjacent to the detection pad is overlapped on the detection pad; in a further preferred embodiment, the blood filtering membrane is a glass cellulose membrane; the combination pad is a glass cellulose membrane; the detection pad is a nitrocellulose membrane; the absorbent paper is made of a material with good water absorption performance, such as a glass fiber material, a cotton pulp material or a mixed material of the glass fiber and the cotton pulp, and the preferred material is the cotton pulp material, or a commercially available absorbent material, such as absorbent paper or an absorbent board, is adopted.
Preferably, in the test strip, a bottom plate is made of a hydrophobic material with certain hardness; further, the bottom plate is a PVC plate.
In a second aspect of the invention, the application of the detection reagent combination of the first aspect in the field of diagnosis of early-onset eclampsia is provided.
Preferably, the application in the field of diagnosis of early eclampsia includes, but is not limited to, any one of the following:
(1) the detection reagent combination is applied to the preparation of an early eclampsia diagnosis product;
(2) acquiring content information of placenta growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1) in a blood sample to be detected based on the detection reagent combination;
(3) and acquiring the content information of the placenta growth factor (PLGF) and the soluble fms-like tyrosine kinase-1 (sFIt-1) in the blood sample of the subject based on the detection reagent combination, and confirming whether the subject belongs to a eclamptic patient.
In the aspect (1) above, the early onset eclampsia diagnostic product is a diagnostic kit, a diagnostic device or a diagnostic platform.
In the aspect (2), the blood sample to be tested is originally derived from a mammal, such as human, monkey, dog, cat, mouse, etc., and a specific example of the non-human mammal is a disease model animal; the purpose of obtaining the content information of the placenta growth factor (PLGF) and the soluble fms-like tyrosine kinase-1 (sFIt-1) also comprises the screening of active drugs.
In the aspect (3), the blood sample is peripheral blood of the subject, and venous blood may be used, and may be selected from a whole blood sample, a serum sample, or a plasma sample.
In the above aspect (3), the determination of whether the subject is a eclamptic patient is made based on the ratio test (sFIt-1/PLGF) of soluble fms-like tyrosine kinase-1 to placental growth factor.
In a third aspect of the present invention, a diagnostic kit for maternal early eclampsia is provided, wherein the kit comprises the combination of the detection reagents for placental growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1) according to the first aspect.
Preferably, in the detection and diagnosis kit, the test strip is protected by a shell.
In a fourth aspect of the present invention, there is provided a method for detecting the content of placenta growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1), wherein the detection method is based on the combination of the detection reagents of the first aspect, to obtain the ratio of the soluble fms-like tyrosine kinase-1 to the placenta growth factor in the blood sample of the subject.
Preferably, the detection method comprises the following specific steps: and dropwise adding the blood sample to be detected onto a blood filtering membrane of the test strip, dropwise adding the washing solution onto the blood filtering membrane for washing after the blood sample is unfolded, and detecting the fluorescence content of a detection line and a quality control line in the detection pad.
Further, the fluorescence content can be detected by a method including, but not limited to, flow, fluorescence microscope, confocal microscope, total internal reflection or two-photon microscope, fluorescence detector, etc., and the fluorescence detection wavelength is 610 nm.
Further, the blood sample unfolding time is 4-6 min.
Further, the washing time is 4-6 min.
The diagnostic kit according to the third aspect of the present invention is used in the same manner as the detection method according to the fourth aspect of the present invention. The placenta growth factor (PLGF) detection method based on the quantum dot fluorescence technology has the following principle:
by adopting a double-antibody sandwich method, the main reaction part is a combination pad and a detection pad (a detection line T1, a detection line T2 and a quality control line are sequentially arranged on a nitrocellulose membrane (NC membrane) of the detection pad): quantum dots are coupled with streptavidin (Q-SA), specific antibodies of an antigen to be detected Ag1 (PLGF) and an antigen to be detected Ag2 (sFIt-1) are coupled with biotin (Bio-Ab1 and Bio-Ab2) respectively, and the three are uniformly mixed and sprayed on a binding pad. After the sample is added, due to the chromatography effect, the quantum dots mark the streptavidin and the biotin-labeled antibody, and move forwards along the nitrocellulose membrane along with the sample solution. The antigens to be detected Ag1 and Ag2 in the sample respectively react with the antigens to form complexes Q-SA-Bio-Ab1-Ag1 and Q-SA-Bio-Ab2-Ag2, and when the complexes pass through the regions of a detection line T1 and a detection line T2 which are solid-phased with capture antibodies, the formed complexes are captured by the capture antibodies and respectively form two peaks T1 and T2. The unbound sample solution moves forward continuously, passes through the quality control line region immobilized with goat anti-mouse IgG (Ab 3), is captured by the quality control line antibody, and forms a C peak, thus completing the quality control calibration reaction. Subsequently, a sample washing solution was added to wash and remove non-specific adsorption.
After washing, putting the detection card into a detection instrument, increasing the fluorescence values T1 and T2 of a detection line area along with the increase of the concentration of PLGF and sFIt-1, and calculating through a standard curve to obtain the content of PLGF and sFIt-1 in a sample to be detected; and further obtaining the ratio of sFIt-1/PLGF, thereby evaluating the incidence probability of eclampsia of the patient.
In a fifth aspect, the present invention provides a method for diagnosing early eclampsia in pregnant and lying-in women, wherein the method comprises obtaining a blood sample from a subject, and detecting placental growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1) in the blood sample by using the detection reagent combination according to the first aspect, the diagnostic kit according to the third aspect, or the detection method according to the fourth aspect.
The beneficial effects of one or more technical schemes are as follows:
1. in the detection reagent combination provided by the invention, on the basis of the existing detection test paper, a streptavidin-biotin amplification system is added, the detection sensitivity is improved, and the detection limit of PLGF is not higher than 15pg/mL and the detection limit of sFIt-1 is not higher than 30 pg/mL.
2. The test paper strip sample adding hole is provided with a blood filtering device: after the blood filtering membrane is pretreated by the buffer solution containing the RBC, the freeze drying mode is adopted, the activity of the RBC is kept to the maximum extent, vacant sites on the blood filtering membrane can be sealed, and the vacant sites are prevented from adsorbing target objects (PLGF and sFIt-1) in a blood sample, so that the purpose of effectively filtering red blood cells is achieved, the detection of the whole blood sample is realized, and the detection results of the whole blood sample, the serum sample and the plasma sample have good consistency.
3. The detection object of the invention, placenta growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1), has high detection requirement on sensitivity, and the detection limit is 15-30 pg/mL. In order to improve the fluorescence signal value of the detection test strip, the streptavidin-biotin amplification system is introduced, so that the fluorescence intensity can be improved by about 4-10 times, but some non-specific adsorption can be introduced at the same time, so that the background is synchronously increased; therefore, in order to realize the synchronous improvement of the detection sensitivity and the specificity, the invention also designs and adds a washing step, after the sample of 50 mu L is added and the display panel is completed for 4-6min, the detergent (containing the surfactant, the protein, the high molecular polymer and the like) is added, the display panel is continued for 4-6min, the effects of removing the non-specific adsorption and closing the sites are achieved, and the detection sensitivity is improved. In addition, the test paper provided by the invention has good stability, and can be transported and stored at normal temperature.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic view of the test strip of the present invention;
wherein, the test paper comprises 1-blood filtering membrane, 2-binding pad, 3-NC membrane, 4-absorbent paper, 5-bottom plate (PVC plate), 6-test line T1 and test lines T2, 7-quality control line.
FIG. 2 is a schematic diagram of the structure of the outer casing of the test strip;
wherein, 8-the sample adding hole, 9-the window.
FIG. 3 is a graph of the PLGF standard curve for the test strip described in example 1;
FIG. 4 is a graph of the sFlt standard curve for the test strip described in example 1.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the technical solution of the present invention will be described in detail below with reference to specific examples and comparative examples.
Example 1
The embodiment provides a quantum dot fluorescence detection method for placenta growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1), the detection method is realized based on the detection test strip shown in fig. 1, the test strip comprises a bottom plate 5, and a chromatographic structure is supported by the bottom plate 5. The chromatographic structure has a blood filtering membrane 1, a combination pad 2, an NC membrane 3 and absorbent paper 4 from the sample adding end to the sample chromatographic direction, the blood filtering membrane 1, the combination pad 2 and the NC membrane 3 are sequentially lapped, and the part of the absorbent paper 4 contacting the NC membrane 3 is lapped on the NC membrane 3.
In this embodiment, the preparation method of the test strip is as follows:
1) preparation of a coating film:
taking an NC film and a PVC plate, pasting the non-sample application surface of the NC film on the set position of the PVC plate, and carrying out scribing and coating of the NC film by the antibody by using a three-dimensional scribing metal spraying instrument.
Taking an anti-PLGF monoclonal antibody with the concentration of 1.2mg/mL, and streaking and coating the anti-PLGF monoclonal antibody at the position of a detection line T1 of an NC membrane; taking an anti-sFIt-1 monoclonal antibody with the concentration of 1.1mg/mL, and marking out the position of a detection line T2 coated on the NC membrane; goat anti-mouse IgG was applied at a concentration of 0.5mg/mL, and the portion of the NC membrane coated with the control line C was streaked. And (3) putting the scribed coating film into an oven, and drying for 8 hours at 37 ℃ to obtain the coating film.
2) Preparation of the bonding pad
(A) Quantum dot labeled streptavidin
a: adding 10 mu L of cadmium selenide quantum dots into 3mL of HEPES buffer solution (pH 7.0) with the ionic strength of 50mM, adding 15 mu L of 20mg/mLEDC and 15 mu L of 20mg/mLNHS, then adding 130 mu L of 5mg/mL streptavidin, and reacting for 4h in a dark place;
b: b, adding casein with the final concentration of 6mg/mL into the quantum dot labeling solution prepared in the step a, stirring and reacting for 30min in a dark place, and sealing;
c: c, transferring the marked mixed solution in the step b to a 200KD ultrafiltration tube for centrifugation at 3000g/min for 15min to ensure that the volume of the centrifuged liquid is 1/10 which is smaller than the original volume; eluting 5 times with 50mM borate buffer (pH 7.4) to ensure that the liquid volume is less than 1/10 of the original volume after each centrifugation;
d: and d, diluting the marked solution obtained in the step c by using a complex solution, and uniformly mixing to obtain the quantum dot marked streptavidin.
(B) Monoclonal antibody of biotin-labeled PLGF and sFIt-1
a: respectively dissolving antibodies to be labeled PLGF and sFIt-1 in a PBS buffer solution to obtain two antibodies to be labeled with the final concentration of 5 mg/mL;
b: weighing biotin and dissolving in ultrapure water to a final concentration of 10 mM;
c: b, respectively adding 30 mu L of biotin solution into 1mL of the two antibody solutions to be labeled in the step a, and stirring and reacting for 30min at room temperature;
d: c, transferring the marked mixed solution in the step c to a 100KD ultrafiltration tube for centrifugation at 3000g/min for 10min to ensure that the volume of the centrifuged liquid is 1/10 which is smaller than the original volume; eluting 2 times with 10mM PBS buffer (pH7.4) to ensure that the volume of liquid after each centrifugation is less than 1/10 of the original volume;
e: and d, diluting the marked solution obtained in the step d by using a complex solution, and uniformly mixing to obtain a biotin-marked PLGF antibody and a biotin-marked sFIt-1 antibody respectively.
(C) Spray pad
And (3) mixing the quantum dot labeled streptavidin prepared in the step (A) with the biotin labeled PLGF antibody and the biotin labeled sFIt-1 antibody prepared in the step (B) according to the ratio of 1: 1: 1, uniformly mixing, and spraying the mixture on a glass fiber membrane by using a three-dimensional scribing metal spraying instrument according to the spraying amount of 3 mu L/cm. And (3) putting the glass fiber membrane subjected to the pad spraying into an oven, and drying for 4 hours at 37 ℃ to obtain the combined pad.
3) Blood filtration membrane pretreatment
a: preparing a pretreatment solution: 0.1M HEPES buffer (pH 7.4) containing 0.9% NaCl, 2% sodium caseinate, 2% PVP-K30, 0.6% S17 and 0.05% Proclin300, 150. mu.g/mL anti-erythrocyte antibody (RBC);
b: b, applying the pretreatment solution prepared in the step a, soaking the blood filtering membrane for 1 hour at room temperature;
c: drying the pretreated blood filtering membrane in the step b by using a freeze dryer to obtain the pretreated blood filtering membrane;
4) assembling:
tearing off adhesive paper at a corresponding position on the bottom plate, adhering absorbent paper at a corresponding position above the NC film, and covering the NC film for 1 mm; adhering the bonding pad below the NC membrane, and covering the bonding pad by 1 mm; and sticking the blood filtration membrane below the combination pad, and covering the combination pad for 2mm to obtain the assembled large plate.
5) Slitting and casing: and (3) cutting by using a microcomputer automatic cutting machine after the width of the test strip is set to obtain the prepared test strip, packaging the test strip into a shell to form the detection card shown in the figure 2, and then sealing the detection card in an aluminum foil bag to obtain the detection card of the single-person placenta growth factor (PLGF) and the soluble fms-like tyrosine kinase-1 (sFIt-1).
The quantum dot fluorescence detection method for placenta growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1) based on the test strip comprises the following specific operation steps:
1) a sample detergent was formulated. According to the optimal formulation, 0.1M HEPES buffer (pH7.4) contains 0.9% NaCl, 5% newborn bovine serum, 1% PEG20000, 0.4% S17 and 0.05% Proclin 300.
2) When the detection method is used for detecting the PLGF and the sFIt-1 in a sample, the detection method comprises the following steps:
a. and (3) sucking 50 mu L of sample to be detected (serum/plasma/whole blood) into the sample adding hole of the detection card, and waiting for 5min of a sample display plate.
b. Adding 50 mu L of sample detergent into the sample adding hole of the detection card, washing, standing for 5min, inserting the detection card into a fluorescence immunoassay analyzer ZF06, clicking 'test' to detect the fluorescence signal intensity of the detection card, respectively representing the fluorescence values of a detection line T1, a detection line T2 and a quality control line by T1, T2 and C, providing the finally obtained detection results in a ratio form (namely respectively taking T1/C, T2/C, T2/T1 as output results), substituting the ratio of the detection results into a standard curve (figures 3 and 4), calculating the concentrations of PLGF and sFIt-1 in the sample to be detected, and simultaneously providing the ratio of sFIt-1/PLGF.
Comparative example 1
In this example, another quantum dot fluorescence detection method of placenta growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1) is provided, wherein the preparation method of the detection card is the same as that of example 1, except that only the serum sample is added and the sample detergent is not added dropwise during the sample detection process.
Comparative example 2
In this example, another quantum dot fluorescence detection method for placenta growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1) is provided, wherein the detection card is prepared in the same manner as in example 1, except that the filter membrane portion of the test strip of the placenta growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1) detection kit is not pretreated.
Comparative example 3
In this embodiment, another quantum dot fluorescence detection method for placenta growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFIt-1) is provided, in which a streptavidin and biotin amplification system is not introduced into a test strip portion of a detection card, i.e., after PLGF and sFIt-1 antibodies are labeled by quantum dots, a spray-on binding pad is uniformly mixed, and then a test strip is assembled.
The synchronous detection comparison of 10 serum samples with lower clinical assigned concentration with the example 1 shows that the detection result of the comparative example 3 has obvious difference and poor linear correlation with the clinical detection result compared with the example 1, which indicates that the sensitivity does not meet the clinical detection requirement under the condition of not introducing an amplification system.
Effect verification
1. Verification of the accuracy of the detection methods in example 1 and comparative examples 1-2
The following clinical samples were obtained from otorhinolaryngology hospitals in Shandong province, wherein the assigned concentrations of the samples were measured by a placenta growth factor detection kit (electrochemiluminescence method) Elecsys PLGF and a soluble fms-like tyrosine kinase-1 detection kit (electrochemiluminescence method) Elecsys sFlt-1, respectively, manufactured by Roche Diagnostics GmbH, and the results of the 20 cases of clinical samples were detected based on the above detection methods, respectively, are shown in the following tables 1-2:
TABLE 1 placental growth factor (PLGF) assay results
TABLE 2 soluble fms-like tyrosine kinase-1 (sFIt-1) assay results
2. Detection sensitivity verification
The technical indexes of the test strip comprise:
1. "accuracy: the relative deviation is not more than +/-10%
Repeatability: CV not exceeding. + -. 10% "
Detection limit: the detection limit of PLGF is not higher than 15pg/mL
The detection limit of sFIt-1 is not higher than 30pg/mL
Linear range: PLGF Linear Range [20, 10000] pg/mL
Linear range [50, 85000] pg/mL for sFIt-1
3. Test strip stability verification
The sample with the known PLGF concentration of 439pg/mL and sFIt-1 concentration of 2200pg/mL is used to perform the placement stability test for different batches of test strips, and 3 batches of test strips are extracted and placed at room temperature, and the test results are shown in the following table 3:
TABLE 3
As can be seen from the test results in Table 3, the deviation of the test value of the test strip in three batches provided by the invention is not more than 5% after the test strip is placed at room temperature for 12 months, and compared with most test strips on the market which need to be stored at low temperature, the verification results show that the test strip and the test card provided by the invention can maintain good stability even at room temperature, and the storage and transportation cost can be obviously reduced.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A detection reagent combination for placenta growth factor and soluble fms-like tyrosine kinase-1 is characterized in that the detection reagent combination comprises a detergent and a test strip for cleaning a blood sample to be detected;
the detergent comprises serum, polyethylene glycol, a nonionic surfactant and a bacteriostatic agent;
the test strip comprises a bottom plate, a chromatographic structure is covered on the bottom plate, a blood filtering membrane, a combination pad, a detection pad and water absorption paper are sequentially arranged on the chromatographic structure according to the sample flowing direction, and the adjacent parts of the above components are connected in an overlapping mode.
2. The assay reagent combination for placental growth factor and soluble fms-like tyrosine kinase-1 according to claim 1, wherein the serum is neonatal bovine serum;
the molecular weight of the polyethylene glycol is 5000-25000, and the polyethylene glycol comprises a polyethylene glycol high polymer or a polyethylene glycol derivative;
the nonionic surfactant is selected from one or a combination of several of S17, S18, S19, S21, S22 and S24;
the bacteriostatic agent is ProClin 300.
3. The test reagent combination for placental growth factor and soluble fms-like tyrosine kinase-1 according to claim 2, wherein said detergent is formulated as follows: 0.07-0.13M HEPES buffer solution contains 0.8-1.0% NaCl, 3-6% newborn bovine serum, 1-2% PEG20000, 0.3-0.6% S17 and 0.03-0.06% Proclin 300.
4. The test reagent combination for placental growth factor and soluble fms-like tyrosine kinase-1 according to claim 1, wherein said hemofilter comprises anti-erythrocyte antibodies for filtering out erythrocytes in a blood sample; the hemofiltration membrane is prepared by spraying RBC-containing pretreatment liquid, and the formula of the pretreatment is as follows: 0.07-0.13M HEPES buffer solution contains 0.8-1.0% NaCl, 1-3% sodium caseinate, 1-3% PVP-K30, 0.4-1.0% S17, 0.03-0.06% Proclin300 and 140-160 μ g/mL anti-erythrocyte antibody.
5. The combination of detection reagents for placental growth factor and soluble fms-like tyrosine kinase-1 according to claim 1, wherein said conjugate pad is coated with a mixture of quantum dot-labeled streptavidin, biotin-labeled PLGF antibody and biotin-labeled sFIt-1 antibody, said mixture being dissolved and diluted in a reconstituted solution and sprayed on said conjugate pad, said reconstituted solution having the following formulation: 0.07-0.13M HEPES buffer solution contains 0.8-1.0% NaCl, 1-3% tyrosine, 4-6% sucrose, 1-2% beta-cyclodextrin, 0.3-0.6% S17 and 0.03-0.06% Proclin 300.
6. The combination of claim 1, wherein the detection pad has a detection line T1, a detection line T2 and a quality control line arranged in sequence along the chromatographic direction, the detection line T1 is coated with PLGF capture antibody, the detection line T2 is coated with sFIt-1 capture antibody, and the quality control line is coated with goat anti-mouse IgG antibody.
7. A diagnostic kit for the early onset of eclampsia in pregnant and lying-in women, comprising the combination of detection reagents for placental growth factor and soluble fms-like tyrosine kinase-1 according to any one of claims 1 to 6, wherein the ratio of soluble fms-like tyrosine kinase-1 to placental growth factor in a blood sample of the subject is obtained based on the combination of detection reagents; in the detection and diagnosis kit, the outer part of the test strip is provided with a shell for protection.
8. The diagnostic kit for maternal early onset eclampsia as claimed in claim 7, wherein the method of use of said kit is as follows: dripping a blood sample to be detected on a blood filtering membrane of the test strip, dripping the washing solution on the blood filtering membrane for washing after the blood sample is unfolded, and detecting the fluorescence content of a detection line and a quality control line in the detection pad;
the fluorescence content detection mode is selected from one of a flow type, a fluorescence microscope, a confocal microscope, a total internal reflection or two-photon microscope and a fluorescence detector, and the fluorescence detection wavelength is 610 nm;
the blood sample is unfolded for 4-6min, and the washing time is 4-6 min.
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